罗尼氏弧菌Vibrio shilonii BY新琼胶酶基因的克隆、序列分析及表达

Cloning, functional analysis and expression of a novel gene encoding agarase from Vibrio shilonii BY

摘要:

【目的】从海洋来源的罗尼氏弧菌菌株BY中克隆得到一个具有琼胶酶活性的新基因，并对其进行重组表达。【方法】对实验室保藏的产琼胶酶菌株BY进行16S rRNA基因序列分析，并构建系统发育树。根据已报道的琼胶酶基因序列的同源性，设计简并引物，利用降落PCR (Touch-down PCR)及染色体步移技术扩增琼胶酶基因序列全长，对基因序列进行生物信息学分析。将目的基因插入pET22a(+)载体，转化大肠杆菌BL21(DE3)，对重组酶进行表达，利用DNS法测定了重组酶的酶活，对该重组琼胶酶酶学性质进行研究。【结果】克隆得到一条新的琼胶酶基因，命名为Vibrio sp. BY (GenBank登录号：AIW39921.1)，Vibrio sp. BY基因序列全长2 232 bp，编码744个氨基酸，理论分子量为85 kD，Vibrio sp. BY的氨基酸序列基因库中与已知的琼胶酶氨基酸序列Vibrio sp. EJY3的相似度为86%。发酵液琼胶酶酶活力为71.73 U/mL，证明表达的蛋白为琼胶酶。酶学性质研究表明重组琼胶酶的最适温度及pH分别为50 °C和7.0，并且具有较好的稳定性。【结论】利用染色体步移技术克隆得到一条新的琼胶酶基因，并在大肠杆菌BL21(DE3)中实现了重组表达，为琼胶酶的应用奠定了基础。

Abstract:

[Objective] The novel gene encoding agarase in a strain of Vibrio shilonii screened from marine was cloned and expressed in E. coli BL21. [Methods] Isolate an agarase producing strain BY, 16S rRNA gene sequence analysis and construct phylogenetic tree. The degenerate primer was designed according to the known agarase gene sequence, touch-down PCR and chromosome walking techniques were used to obtain agarase full-length gene sequence. After being assembled, the full-length of agarase gene was inserted into expression plasmid pET22a(+), then transformed into E. coli BL21(DE3). Activity of recombinant enzyme was determined utilizing DNS method, study the enzymatic properties of the recombinant enzyme. [Results] A new gene Vibrio sp. BY was cloned from strain BY genomic. The full length of Vibrio sp. BY consists of a 2 232 bp, encoding 744 amino acids, with a putative molecular mass of 85 kD, the amino acids sequence of agarase Vibrio sp. BY shared 86% identity with the agarase of Vibrio sp. EJY3. The activity of agarase was 71.73 U/mL, which indicated that Vibrio sp. BY was an agarase gene. The optimum temperature and pH for the recombinant activity was at 50 °C and 7.0, have a high agarase stability. [Conclusion] A new agarase gene was cloned by chromosome walking techniques, realized recombinant in E. coli BL21(DE3), which lays a good foundation for the research on application of agarase.